Identification of a Spike-Specific CD8+ T-Cell Epitope Following Vaccination Against the Middle East Respiratory Syndrome Coronavirus in Humans

Abstract Licensed vaccines against the Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging pathogen of concern, are lacking. The modified vaccinia virus Ankara vector-based vaccine MVA-MERS-S, expressing the MERS-CoV-spike glycoprotein (MERS-S), is one of 3 candidate vaccines in clinical development and elicits robust humoral and cellular immunity. Here, we identified for the first time a MERS-S–specific CD8+ T-cell epitope in an HLA-A*03:01/HLA-B*35:01-positive vaccinee using a screening assay, intracellular cytokine staining, and in silico epitope prediction. As evidence from MERS-CoV infection suggests a protective role of long-lasting CD8+ T-cell responses, the identification of epitopes will facilitate longitudinal analyses of vaccine-induced T-cell immunity.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging zoonotic pathogen.It belongs to the Betacoronavirus genus, which includes the endemic human coronaviruses (HCoV-OC43 and HCoV-HKU1) and the epidemic SARS-CoV and SARS-CoV-2 [1,2].Infection with MERS-CoV leads to MERS, a primarily respiratory disease with a case fatality rate of approximately 35% [1,2].Since 2012, >2000 cases have been reported, mostly on the Arabian Peninsula [1,2].Dromedary camels serve as a natural reservoir for MERS-CoV, harboring a constant risk of spillover to humans and viral evolution [1,2].Due to this high epidemic potential, MERS-CoV is listed by the World Health Organization as a priority pathogen of concern for epidemic preparedness measures, such as vaccine development [1,2].
The current vaccine candidates are predominantly based on the MERS-CoV spike protein (MERS-S), a highly immunogenic surface protein, that facilitates the binding (S1 subunit) and fusion (S2 subunit) of MERS-CoV with its host cells [2].One of the 3 MERS vaccine candidates in clinical development is the viral vector vaccine MVA-MERS-S, based on the modified vaccinia virus Ankara (MVA) expressing the fulllength MERS-S [3].In a first-in-human phase 1 clinical trial (ClinicalTrials.govidentifier NCT03615911), we previously demonstrated the induction of robust humoral and cellular immunity after 2 MVA-MERS-S vaccinations.MERS-S-specific T-cell responses were detectable in 87% of vaccinees (n = 20/23) upon stimulation with MERS-S-specific overlapping peptide (OLP) pools [4].Interestingly, in some vaccinees T-cell responses were already measurable after the first dose and persisted for a longer time compared to immunoglobulin G levels [4].
There is still a limited understanding of T-cell immunity elicited by MERS-CoV infection or vaccination, but evidence from MERS survivor studies suggests a protective and longlasting role [5][6][7].In particular, CD8 + T cells were detectable even in patients with mild or asymptomatic disease, in the absence of relevant antibody titers [5].MERS-specific CD8 + T cells may therefore be important for monitoring immunogenicity of MERS vaccines longitudinally.MERS-CoV-specific T-cell epitopes, however, remain mostly unknown, hindering the development of tools, such as tetramers, for the detection, isolation, and characterization of antigen-specific T cells.While several MERS-S-specific CD4 + T-cell epitopes have been described in MERS survivors [5], MERS-S-specific CD8 + T-cell epitopes have only been predicted in silico [8] and to date have not been identified in humans.The Journal of Infectious Diseases

B R I E F R E P O R T
To address these constraints, the aim of this study was to identify MERS-S-specific CD8 + T-cell epitopes after vaccination with the viral vector vaccine candidate MVA-MERS-S.We analyzed the T-cell response to single overlapping peptides covering MERS-S in the vaccinee with the highest overall T-cell response in the phase 1 clinical study and identified the first MERS-Sspecific CD8 + T-cell epitope candidate after MERS vaccination.

METHODS
The vaccinee described here was part of a first-in-human phase 1 clinical trial of the vaccine candidate MVA-MERS-S (NCT03615911, published [4]).The trial consisted of a homologous prime-boost scheme of 1 × 10 7 or 1 × 10 8 plaqueforming units/mL of MVA-MERS-S on days 0 and 28.The study was approved by the competent national authority (Paul Ehrlich Institute) and the Ethics Committee of the Hamburg Medical Association.Informed consent was obtained from all participants.The study was performed in accordance with the Declaration of Helsinki (2013).For immunogenicity analysis, peripheral blood mononuclear cells (PBMCs) were collected on days 7, 14, and 28 after the second vaccination (V2D7, V2D14, and V2D28, respectively).HLA typing was performed using commercially available A and B locus kits (LABType SSO Typing Test, ONE LAMDA) according to the manufacturer's instructions.
Multiple sequence alignment revealed that P19 is specific to the MERS-S and not conserved in human betacoronaviruses (Supplementary Table 3).Next, we performed in silico epitope prediction of the complete MERS-S sequence for HLA-A*03:01 and HLA-B*35:01, the HLA-class I alleles for which the vaccinee was homozygous.Notably, an 11-mer (FPYQGDHGDMY) and a 13-mer epitope (FPYQGDHGDMYVY) within the P19 peptide were among the top 5 predicted HLA-B*35:01 MERS-S epitopes with the highest affinity (percentile rank = 0.01; Supplementary Table 4).One 9-mer epitope was identified in the HLA-A*03:01 prediction analysis, but with a lower affinity (percentile rank = 0.38; Supplementary Table 5).
By IFN-γ ELISpot we could show that the only other HLA-B*35:01 positive vaccinee of the clinical trial also responded to P19 (Supplementary Figure 4).Notably, P19 was not recognized by the 2 vaccinees who carried the HLA-A*03:01 but not the HLA-B*35:01 allele.

DISCUSSION
The induction of functional and long-lasting immune responses is fundamental for successful vaccine design.The protective and durable nature of CD8 + T cells in MERS survivor studies makes them interesting targets for monitoring vaccine immunogenicity.Using an epitope-mapping approach, we identified a novel MERS-S-specific CD8 + T-cell epitope candidate (P19) in an individual vaccinated with MVA-MERS-S and could validate this finding in one further HLA-A*03:01/HLA-B*35:01-positive vaccinee.We observed a higher P19-specific T-cell response compared to all other peptides of the M1 pool and the M2-M5 pools, suggesting the potential immunodominance of this peptide within MERS-S.
P19-specific T cells were predominantly polyfunctional CD8 + T cells, and expressed CD107a, a marker of cytotoxicity.Polyfunctional CD8 + T cells can exert multiple effector functions and are associated with protection from disease.Interestingly, most P19-specific cells were T emras , with an increasing frequency from V2D14 to V2D28.CD8 + T emras have been associated with protection against dengue and symptomatic H1N1 influenza and shown to be long lasting following dengue vaccination [10][11].While the contribution of MERS-specific T emras to protection remains elusive, previous studies have shown that memory T-cell responses, including IFN-γ-secreting T emras, were detectable up to 6.9 years after infection [5,7], suggesting that they may drive the maintenance of immune memory.
A beneficial characteristic of P19 for immunogenicity monitoring after MERS vaccination is its location in the N-terminal domain of the S1 subunit, outside the RBD, a region less prone to mutations or cross-reactivity.While the RBD is known to be highly immunogenic, mutations in the RBD have been implicated in the emergence of immune evasion in variants of SARS-CoV-2 [12].These mutations may not only evade the humoral immune response but may also impact potential T-cell epitopes.Additionally, a report of the ChAdOx1-MERS vaccine clinical trial suggests that the RBD may have a role in T-cell cross-reactivity, given that preexisting T-cell responses detectable in a small number of vaccines were predominately directed against the RBD [13].A phylogenetic analysis of 484 MERS-CoV isolates from humans and camels revealed no mutations in the P19 sequence [14].Furthermore, our sequence alignment showed that P19 is not conserved among HCoVs.Taken together, the detected T-cell epitope mapping to a less mutagenic location of the spike with a highly specific sequence for MERS-CoV seems favorable for the longitudinal assessment of anti-MERS-CoV vaccine responses.This is particularly important given that preexisting immune responses to endemic HCoVs and SARS-CoV-2 are now highly prevalent and may interfere with immune monitoring during upcoming MERS vaccine trials.
In silico prediction revealed that P19 likely encompasses an HLA-B*35:01-restricted epitope.Notably, the allele frequency for HLA-B*35:01 ranges from 1.4% to 13.5% worldwide.HLA-B*35:01 is present in Germany (5.8%) but also in Saudi Arabia (3.0%), where MERS-CoV is endemic [15].However, further studies are needed to identify the optimal epitope sequence, as MHC-I binding favors amino acid lengths of 9-11.A limitation of this study is that analyses were only performed in a limited number of vaccinees.However, we could show that P19 is recognized by all HLA-B*35:01-positive vaccinees of our phase 1a clinical trial.In conclusion, the identified CD8 + T-cell epitope may facilitate the implementation of tetramers to provide novel insights into the role of CD8 + T cells in anti-MERS-CoV immunity, which may further accelerate MERS-specific immune monitoring and the development of more efficacious vaccine candidates.